Project Summary Lynch syndrome (LS) is a hereditary disease that predisposes patients to colorectal, endometrial, ovarian and other cancers. Definitive diagnosis of LS depends on detection of a deleterious, germline mutation in one of the DNA mismatch repair (MMR) genes. A problem arises for clinicians and genetic counselors, however, when the sequencing reveals a variation, such as a missense mutation, whose effect on gene function is not immediately obvious. These variants of uncertain significance (VUS) cannot be used to confirm nor rule out LS resulting in uncertainty about how to manage the patient and their family members. Laboratory studies to determine whether a VUS disrupts protein function has become one important part of a strategy for determining their relevance to disease. These functional studies, however, can be tedious and as such, only limited progress has been made in characterizing the functional effects of identified variants over the past 20 years. What is needed is a way to rapidly screen thousands of MMR gene variants for functional consequences. CRISPR-Cas9 mediated gene editing provides an excellent means for creating variants in human cell culture models for functional testing. We propose to combine CRISPR-Cas9 gene editing in human pluripotent stem cells (PSCs) with large-scale genomics approaches in order to create every possible single nucleotide variant (SNV) in the MMR gene MSH2. Taking advantage of the fact that MMR-deficient cells are resistant to the lethal effects of SN1 DNA alkylating agents, we will perform a screen to identify those variants in MSH2 which disrupt MMR function via the following aims: 1) Test the effect of all possible SNVs in representative exons of MSH2 on MMR function. We will use CRISPR-Cas9-mediated gene editing using a pool of homology-directed repair templates to alter each nucleotide position in exons 6 and 11 of MSH2. We will perform next-generation DNA sequencing of the targeted region to determine the number of cells carrying each variant. We will then treat the cell population with a DNA alkylating agent and sequence again to identify which variants are enriched, indicative of MMR disruption. 2) Confirm the functional effects of individual missense mutants of MSH2. We will ?knock-in? individual missense VUS into the endogenous MSH2 locus in hESCs and examine their effects on various MMR functions including protein stabilization, DNA repair and alkylation damage sensitivity and compare their effects to that predicted in Aim 1. This proposal will set the stage for future studies in which we will be able to test the functional effect of all possible SNVs in MSH2 as well as the other major MMR genes. These studies will assist in the disease significance classification for all MMR gene VUS which can be used by clinicians and genetic counselors world-wide to assist in managing and preventing cancer in patients and their families.